DNA replication plays a central role in life. Using specialized enzyme—DNA polymerases, and some auxiliary proteins, cells and viruses are able to precisely copy the genetic information of the primary organism to create its multiple copies.
Polynucleotide replication plays a crucial role in many in vitro nucleic acid diagnostic and research tools. For many years polynucleotide replication has been used as a key reaction in polynucleotide sequence analysis that include a traditional gel-based sequence analysis (Sanger method) and a more recent highly parallel polynucleotide sequencing technology (for example, Solexa platform).
Polynucleotide replication is also a part of several current methods of genotyping. It is routinely used for labeling a polynucleotide with fluorescent dyes for microarray analysis, and it is used for in situ detection using Primed In Situ Labeling (PRINS).
However, one of the most important applications of polynucleotide replication is amplification. Examples include but not limited to Polymerase Chain Reaction (PCR), Strand Displacement Amplification (SDA), Rolling Circle Amplification (RCA), Helicase-Dependent Amplification (HDA), Single Primer Isothermal Amplification (SPIA), etc.
Many of polynucleotide amplification methods rely on the primer extension reaction repeated multiple times. To achieve this goal it is important to dissociate the polynucleotide polymerization product from its nucleic acid template. In PCR such dissociation is achieved by heating the duplex polynucleotide product repeatedly up to 95° C. In other methods, such as SDA, HDA and SPIA, dissociation of the extended product is facilitated by additional enzymes such as restriction endonuclease or nickase (SDA), RNase H (SPIA), or helicase (HDA).
Rolling circle amplification (RCA) is a unique example of primer extension reaction where multiple rounds of replication of a circular polynucleotide template can be achieved without thermal denaturation of double stranded polynucleotides or additional enzymes. The product of RCA is a repetitive sequence (cA)n where A is a sequence of the circular template and cA is its complement. However, this technique is limited to the synthesis of only this type of extension product. RCA cannot be used to synthesize, for example, more complex sequence combinations like cABC or (cABC)n, wherein each of A, B, and C is a unique sequence and cABC is the complement of ABC. While this limitation of RCA to synthesize molecules like (cABC)n is likely due to the fact that the production of the circular polynucleotide templates used in this technique requires special circularization and purification procedures, which consequently limits the size of the template, and, in turn, limits the size of the extension product, the limitation to synthesize a non-repetitive sequence like cABC comes from difficulty to control circular DNA replication. Another drawback of RCA is that only some of the target polynucleotides are elongated, while others are left unreacted (and non-extended).
In view of the foregoing, there exists a need in the art for efficient methods of primer extension where (i) multiple rounds of replication of a polynucleotide template are achieved without thermal denaturation of double stranded polynucleotides and without the use of additional enzymes for dissociation of the template from the extension product, (ii) the type (e.g., complexity, size) of the extension products synthesized are not restricted by complicated template production procedures, and (iii) each of the intended target molecules are elongated in an evenly distributed fashion.